Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.     

Transblot identification of biotin-containing proteins in rat liver

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.     

NK-like cytotoxicity of allospecific gamma delta TCR+ T cell clones]

We recently generated a series of human alloantigen-specific, CD3+, gamma delta- TCR+ clones by stimulating CD3+, CD4-, CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). These clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Most but not all of these clones express the NK cell associated marker, CD57, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not NK-resistant line, Raji. Gamma delta clones which lacked expression of CD57 had no detectable NK activity. The allospecific cytotoxicity of CD57+ and CD57- clones was inhibited by mAb to CD3 or the TCR delta- chain. In contrast, the NK-like activity of the CD57+ clones was enhanced by these antibodies over a wide range of antibody concentration. An HLA class I framework-specific mAb had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the gamma delta- TCR+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+, TCR- gamma delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Cloning and sequencing of a ligninase gene from a lignindegrading basidiomycete,Phanerochaete chrysosporium

Summary A ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3 and 5 intron boundaries. The putative eukaryotic regulatory sequences, i.e. CAAT and TATA box-like sequences, are present in the 5 flanking region.     

The amplified long genomic sequence (ALGS) located in the centromeric regions of mouse chromosomes.

We reported previously that the haploid genome of standard strains of laboratory mice contains approximately 70 copies of an amplified long genomic sequence, designated ALGS, that includes a retroposon of the gene for elongation factor 2 (MER). The length of each repeating unit is more than 60 kb, and the sequence of the unit is highly conserved among the repeats. In the present study, Southern blot analysis of the genomes of wild rodents demonstrated that the ALGS is present in all subspecies of Mus musculus and is abundant in M. spicilegus, whereas it is absent in M. spretus as well as in Rattus and other closely related genera. This result indicates that the amplification occurred after the species differentiation with the genus Mus and at least prior to the differentiation of subspecies of M. musculus. To locate chromosomal positions of the ALGS, in situ hybridization was carried out with laboratory strains and wild mice. It appears that the ALGS is located in the centromeric regions of most chromosomes in laboratory mice, M. musculus and M. spicilegus, whereas no positive signals were observed with M. spretus, in accordance with the results from the Southern blotting analysis.     

Analysis of strongly acidic amino acids by the conventional amino acid analyzer: application to determination of protein-bound cysteine and glutathione

A rapid analysis method of strongly acidic amino acids and related compounds by a simple modification of an existing amino acid analyzer is presented. In this method, an anion-exchanger column (2.6 X 150 mm) packed with Hitachi 3013-N resin was developed with 0.2 M citric acid. Complete separation of phosphothreonine, phosphoserine, phosphotyrosine, cysteic acid, homocysteic acid, and glutathionesulfonic acid was achieved within 35 min, with no regeneration of the column being required. Tyrosine-O-sulfate was analyzed by the same column using 2 M sodium acetate buffer, pH 5.5. Performic acid oxidation of a variety of proteins and direct analysis of the products by this system successfully detected cysteine, homocysteine, and/or glutathione bound to proteins through disulfide bonds. This suggest the potential use of the method for analysis of the states of protein thiol groups, especially those of clinically significant mutant proteins where mutation of arginine to cysteine is rather frequently recognized.     

Isolation and partial characterization of an ion channel protein from human sperm membranes

Human sperm cells were fractionated and plasma membrane proteins were separated by molecular gel sieving chromatography (Sephacryl S-200 followed by HPLC). A pore-forming protein was extracted from sperm cell membranes. The partially purified protein migrated with Mr 100,000-110,000, as determined by molecular sieving gel chromatography, and with a Mr 90,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The channel activity was also extracted with Triton X-114, suggesting a hydrophobic nature for this protein. This protein was incorporated into planar lipid bilayers, resulting in the formation of voltage-dependent ion channels. Single channel fluctuations of 130 pS/unit in 0.1 M NaCl were resolved; however, channels preferentially aggregated in triplets having an open state life-time that persisted for several seconds. The channels studied here were more selective for monovalent cations than anions, but also showed some permeability to anions and larger electrolytes, suggesting a large functional pore diameter. The role of this sperm channel in normal sperm physiology and/or fertilization is presently unclear.     

Suppression of natural killer cell activity by human seminal plasma in vitro: identification of 19-OH-PGE as the suppressor factor

Human seminal plasma (SP) was found to contain a potent suppressor of human natural killer (NK) cell cytotoxic activity against the K562 erythroleukemia target in vitro. Pooled and filter-sterilized SP reduced the NK cell activity of normal blood donors in a concentration-dependent manner; strong reduction of target cell lysis was observed at a final dilution of 1/400. Possible toxic effects of SP on NK cells were ruled out, because donor leukocytes incubated in a final SP dilution of 1/100 remained greater than 99 percent viable as determined by trypan blue exclusion. SP did not affect the lysability of the tumor targets, but suppressed the cytotoxic activity of the effector leukocytes, an effect that was reversed after washing the leukocytes free of the SP. The suppressive action of SP was retained after heating to 95 degrees C for 10 min, but was removed after adsorption with activated charcoal (Norit-A). Lipids extracted from SP were tested for suppression of NK cell cytotoxic activity, and the active principle was identified with the acidic lipid fraction. Components of the acidic lipid fraction of a single freshly obtained SP sample were separated by high pressure liquid chromatography, and suppression of NK cell cytotoxic activity was found to be associated with the predominant prostaglandins (PG) in this fraction, 19-OH-PGE1 and 19-OH-PGE2. Suppression of cellular immune functions by SP has been described; however, the identity of the suppressor factor (or factors) is unknown. The 19-OH-PGE are present in high concentration in primate semen, and may minimize immunologic sensitization to sperm in females after insemination. In addition, these compounds may suppress NK cell antitumor and antiviral activities in the vagina, as well as in their tissues of origin.     

Enhancement of ATPase Activity by a Lipid Peroxide of Arachidonic Acid in Rat Brain Microvessels

The effects of 15-hydroperoxyarachidonic acid (15-HPAA) on Na+, K+- and Mg+-ATPase activities in the blood-brain barrier (BBB) were examined using rat brain microvessels (MV). 15-HPAA markedly stimulated these ATPase activities in MV at low concentrations whereas the synaptosomal Na+, K+-ATPase activity was inhibited in a dose-dependent manner. Further neurochemical analysis revealed that this stimulatory effect of 15-HPAA in MV was not due to a simple detergent-like action of the compound on the membranes but rather to stimulation of the phospholipase A2 and lipoxygenase activity within MV. In addition, it was shown that free radical reactions were involved in the mechanism. Since such anti-edema drugs as 1,2-bis(nicotinamido)propane were proved to be potent suppressors of the enhanced ATPase activity, further speculations on the role of this effect for ischemic brain edema are offered.